Properties of the internal invertase of yeast, Saccharomyces cerevisiae

Abstract

1. 1. Internal yeast invertase (β- d-fructofuranoside fructohydrolase, EC 3.2.1.26) has been isolated and purified from fresh, sonicated Saccharomyces cerevisiae. The specific activity of the pure enzyme was 3128 μmoles sucrose hydrolyzed per ml per min per mg of protein at 25°. The enzyme has an elution volume 1.6 times the void volume on Sephadex G-200 at pH 7.5. 2. 2. A specific, reversible inhibitor effect of cations, notably NH 4 + was found. 3. 3. The internal yeast invertase is inhibited by iodine and the activity is regenerated by mercaptoethanol in an identical way to external invertase. The internal enzyme is also inhibited by cyanogen bromide in a two-step reaction. There is a fast, pH-dependent inhibition and a slow inhibition with a non-ionizing species; the rate of the latter reaction is affected slightly by pH. The slow reaction had a pseudo-first-order rate constant between 22 and 75·10 −6 sec −1 between pH 4.0 and pH 8.0, respectively. K 2PtCl 4 inhibits internal yeast invertase non-competitively with a dissociation constant of 0.025 M at pH 4.9. 4. 4. Internal iodine-invertase has a V which is 40% the value for the native enzyme; the K m of iodine invertase is 1.5 times the K m of the native enzyme. 5. 5. Internal invertase has transferring activity. Internal iodine-invertase has qualitatively the same activity. We were unable to show the formation of aniline fructosides with internal invertase, although we demonstrated such synthesis with external yeast invertase. External invertase can transfer fructose to methanol and to ethanol but not to glycollic acid. It can synthesize the O- fructoside with ethanol but not the analogous S- fructoside with ethanethiol. 6. 6. We conclude that the inhibition by iodine is due to the reversible oxidation of a reactive methionine sulphur atom. The slow inhibition by cyanogen bromide is also due to reaction at a methionine sulphur atom; but these are different methionine residues and, consequently, we suggest that there are two methionine residues at or near the active site of yeast invertase. 7. 7.The data presented are consistent with the mechanism of action proposed for external invertase by A. Waheed and S. Shall ( Biochim. Biophys. Acta, 242 (1971) 172) and we suggest that the external and internal yeast invertases have identical mechanisms of reaction.